The mu opioid receptor, the type most closely associated with both analgesia and behavioral reinforcement, is subject to a variety of factors that regulate its effectiveness in eliciting a cellular response. Among these are the structure of the receptor itself, its density in the cell membrane, and its proximity to coupling proteins. In addition, these factors are all linked by dynamic interactions whose timing, only partially understood at present, can have important influence on the performance of a drug. During the past year we have investigated these factors and dynamics in two ways. The first employed confocal microscopy and a high affinity fluorescent antagonists, "FNAL" AND "6-BNX". The second is the ongoing study of the irreversible antagonist beta-FNA to determine whether a mutant receptor, H297Q, remains capable of covalent modification by the drug and how its response to the drug differs from that of the wild-type receptor. The results of the two programs follow: (1) Confocal microscopy is not only useful to localize probes at a subcellular level, but it can also quantitate them on living cells in real time, as we showed in 2000. In this respect it is comparable to radioligand binding. Unlike radioligand binding, however, we showed this year that the optical sectioning capability of confocal microscopy allows one to clearly distinguish kinetically the binding of drugs to the upper surface of a monolayer culture of cells from the binding to a level only 8 micrometers deeper, and that the influence of this "unstirred layer" depends upon the drug used. This work demonstrates the uniqueness of confocal microscopy, as no other technique we know of can resolve these spacial and kinetic differences. In related confocal microscopic studies, we demonstrated the effects of opioids on intracellular concentrations of both calcium and sodium, revealed by fluorescent probes of these ions. (2) The kinetic studies with beta-FNA, completed this year, raised two important questions that we subsequently pursued. The first was whether sodium, well known to enhance the affinities of agonists binding to isolated cell membranes, likewise affects transduction in whole, living cells. Using Xenopus oocytes expressing wild-type mu opioid receptors and replacing sodium with choline, we showed that there was a small increase in maximum response in the absence of sodium, but that, in contrast to the binding work, EC50 was only marginally altered if at all. However in the mutant receptor H297Q, which recognizes classical alkaloid antagonists as partial agonists, the effect of sodium was more exaggerated. This is a clue into the elusive question of partial agonism that we are still working to understand.